A protein, Tm1631 from the hyperthermophilic organism Thermotoga maritima belongs to a domain of unknown function protein family. It was predicted that Tm1631 binds with the DNA and that the Tm1631–DNA complex is an endonuclease repair system with a DNA repair function (Konc et al. PLoS Comput Biol 9(11): e1003341, 2013
Trang 1Ogrizek et al Chemistry Central Journal (2016) 10:41
DOI 10.1186/s13065-016-0188-6
RESEARCH ARTICLE
Role of magnesium ions in the reaction
mechanism at the interface between Tm1631 protein and its DNA ligand
Mitja Ogrizek1, Janez Konc1,2,3, Urban Bren1,2,3, Milan Hodošček1* and Dušanka Janežič3*
Abstract
A protein, Tm1631 from the hyperthermophilic organism Thermotoga maritima belongs to a domain of unknown
function protein family It was predicted that Tm1631 binds with the DNA and that the Tm1631–DNA complex is an endonuclease repair system with a DNA repair function (Konc et al PLoS Comput Biol 9(11): e1003341, 2013) We
observed that the severely bent, strained DNA binds to the protein for the entire 90 ns of classical molecular dynamics (MD) performed; we could observe no significant changes in the most distorted region of the DNA, where the cleav-age of phosphodiester bond occurs In this article, we modeled the reaction mechanism at the interface between Tm1631 and its proposed ligand, the DNA molecule, focusing on cleavage of the phosphodiester bond After addi-tion of two Mg2+ ions to the reaction center and extension of classical MD by 50 ns (totaling 140 ns), the DNA ligand stayed bolted to the protein Results from density functional theory quantum mechanics/molecular mechanics (QM/ MM) calculations suggest that the reaction is analogous to known endonuclease mechanisms: an enzyme reaction mechanism with two Mg2+ ions in the reaction center and a pentacovalent intermediate The minimum energy
pathway profile shows that the phosphodiester bond cleavage step of the reaction is kinetically controlled and not thermodynamically because of a lack of any energy barrier above the accuracy of the energy profile calculation The role of ions is shown by comparing the results with the reaction mechanisms in the absence of the Mg2+ ions where there is a significantly higher reaction barrier than in the presence of the Mg2+ ions
Keywords: DUF72, Tm1631, QM/MM, Unknown function, ProBiS, CHARMM, GAMESS, Thermotoga maritima
© 2016 The Author(s) This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/ publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated.
Background
Tm1631 is a member of the domain of unknown function
72 (DUF) family in the Protein family (Pfam) database, a
protein domain that has no characterized function [1]
Protein function can only be unambiguously determined
experimentally, but in case of a new protein with no
com-putationally predicted putative function it is difficult to
choose the correct experiment New procedures are
nec-essary to facilitate this research and improve
determina-tion of funcdetermina-tion for all the DUF proteins A new approach
to this problem has been developed by our group and was
described in a previous report to predict the function of the Tm1631 protein [2]
In an earlier paper [2], we used the binding site com-parison capability of the ProBiS algorithm [3 4] to predict the binding site in the Tm1631 protein and to speculate
on the nature of the DNA ligand that could bind to the Tm1631 protein The Tm1631 protein was predicted to
be analogous to endonuclease IV despite sharing <10 % sequence identity, and the proposed Tm1631–DNA complex was subjected to 90 ns long classical MD, using the CHARMM simulation package [5] This resulted in structures of the Tm1631–DNA complex that are used
in the QM/MM study reported in this paper, where we attempted to investigate the catalytic mechanism of the reaction between Tm1631 and DNA It was of especial interest in this connection to determine how Mg2+ ions act in this DNA binding site (Fig. 1) Endonuclease repair
Open Access
*Correspondence: milan@cmm.ki.si; dusanka.janezic@upr.si
1 National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia
3 Faculty of Mathematics, Natural Sciences and Information Technologies,
University of Primorska, Glagoljaška 8, 6000 Koper, Slovenia
Full list of author information is available at the end of the article
Trang 2mechanism is an important mechanism that allows
organisms to escape DNA damage and plays a major
role in the prevention of cancer in higher organisms [6]
Endonucleases cleave phosphodiester bond of DNA at
the damaged site and create a nick in the phosphodiester
backbone that is recognized by further repair enzymes in
the base excision repair pathway The endonuclease
cata-lytic mechanism is thought to involve a hydroxide ion
derived from water, which forms a bond with phosphorus
of the DNA and induces cleavage of the phosphodiester
bond
Endonucleases require one, two or three divalent metal ions, such as Mn2+ or Mg2+ in the catalytic site [7–13] It is believed that in thermal environment, such
as the one Thermotoga maritima lives in at temperatures
around 80 °C, the most suitable metal ion for this kind
of system is Mg2+ [14] However, Tm1631 crystal struc-ture (PDB: 1VPQ) does not contain any metal ions in the predicted binding site On the other hand, there are rela-tively few crystallographically characterised magnesium binding sites [15] For some binding sites, a metal ion is observed in the binding site but which metal ion it is and
Fig 1 The proposed reaction mechanism for Tm1631–DNA complex a Reaction area is encircled The damaged nucleotide abasic dideoxyribose
(3DR), which has no base (abasic site), is on position 7 of the 15 base pair long DNA chain b Structure-based reaction mechanism of
phosphodies-ter bond cleavage The abasic site on the DNA is coordinated by the two Mg 2+ ions, of which one also attacks the hydroxyl nucleophile (left panel)
Pentacovalent transition state [ 20 – 22] (middle panel) collapses, which leads to the cleavage of the scissile phosphodiester P-O3′ bond, with the
transition state and the O3′ leaving group stabilized by the metal ion and inversion of the phosphate configuration (right panel)
Trang 3Page 3 of 9
Ogrizek et al Chemistry Central Journal (2016) 10:41
how many of them are needed for the catalytic activity
remains undetermined [16–19]
Here, we report the results of theoretical QM/MM
studies of the reaction mechanism for the Tm1631
pro-tein We postulated a catalytic mechanism with two Mg2+
ions that resembles the one of the apurinic/apyrimidinic
(AP) endonuclease enzyme presented by Mol et al [21]
We tested the system with and without the ions, and
found that the energetically most favourable pathway of
the phosphodiester bond cleavage catalysed by Tm1631
requires presence of Mg2+ ions In the proposed catalytic
mechanism (Fig. 1b), Lys73 of the Tm1631 should be in a
deprotonated state before water ionizes One of the Mg2+
ions attacks the water molecule, and subsequently water
ionizes, the proton forms a bond with Lys73 and the OH
group forms a bond with the phosphorus atom The
tran-sition state then collapses leading to cleavage of the
phos-phodiester P-O3′ bond, while the O3′ leaving group is
stabilized by the second Mg2+ ion Our findings suggest
that the catalytic mechanism of Tm1631 requires Mg2+
ions and is similar to known Steitz’s mechanism [23]
Experimental and computational studies [24, 25] point
out that the cleavage of the phosphodiester bond occurs
via SN2 nucleophilic substitution in three steps (Fig. 1b)
explained in work by Sgrignani et al [26] and Yang et al
[27]
Methods
Calculations were based on the crystal structure of
pro-tein Tm1631, (PDB: 1VPQ) and the DNA chains from
another crystal structure (PDB: 2NQJ)
System setup and MD
The structures used in this work were previously
equili-brated by classical MD simulation Since there is no
crystal structure of Tm1631 with DNA available in the
Protein Data Bank (PDB), we used as the starting
struc-ture for this study the predicted Tm1631–DNA
com-plex after 90 ns of classical MD, which was performed in
our previous study [2] To validate this starting complex
structure, we plotted its all-atom, protein Tm1631 and
DNA root-mean-square deviations (RMSDs) (compared
to the first snapshot at 0 ns of classical MD) dependence
against the simulation time, which showed that in the last
20 ns of simulation the RMSDs have reached a plateau,
suggesting that the starting structure is well equilibrated
(Additional file 1: Figure S1)
In order to obtain good reactant and product structures
we probed different Mg2+ ion and water molecules
posi-tions In this search we required that Mg2+ initially
coor-dinates with six oxygen atoms (Additional file 1: Table
S1; Distances to Mg12+ and Mg22+), and with the distant
environment atoms in the same positions for both, the
reactant and product conformations The final energy minimization procedures were performed without any constraints or restraints and we were able to obtain suit-able starting positions for the reaction mechanism stud-ies with the above mentioned propertstud-ies
Model building and QM/MM simulation
The starting structure of our simulation was the Tm1631–DNA complex after 90 ns of classical MD simu-lation We replaced two water molecules with two Mg2+
ions and then minimized the system A minimized struc-ture with Mg2+ ions was further optimized at quantum mechanics/molecular mechanics (QM/MM) level using the CHARMM software package The CHARMM force field parameters were used to describe the molecular mechanics (MM) part, while the quantum mechanics (QM) region (42 atoms in total, including both Mg2+ ions (Fig. 2a, b) was treated at the density functional theory (DFT) level using the B3LYP functional and the 6–31G* basis set We used the Replica path (RPATh) method to divide the system into 16 structures equidistantly apart in the RMSD space between the reactants and products and minimized each obtained structure using 3000 steps of adopted basis Newton–Raphson (ABNR) minimization After minimization we checked the distances between
Mg12+ and Mg22+ ions, which were both around 4 Å Both Mg2+ ions were coordinated with 6 oxygen atoms (reactants, Fig. 2c; product, Fig. 2d), and the distances between coordinated atoms and both Mg2+ ions were approximately 2 Å
QM/MM
molecular mechanics part of the calculations, we used CHARMM force field version 36 The ab initio DFT cal-culations were performed using the general atomic and molecular electronic structure system (GAMESS) [29] software package, interfaced to the CHARMM program
We used the B3LYP/6–31G* level of theory, which is implemented in the GAMESS program The ABNR mini-mization algorithm was used for energy minimini-mizations Molecular mechanics calculations were performed with
a constant dielectric of ε = 1 using a classical force shift method and a cut-off distance of 12 Å Molecular graphic images were produced using the VMD software package [30] All ab initio QM/MM and QM calculations were carried out on the CROW clusters at the National Insti-tute of Chemistry in Ljubljana [31]
RPATh
Chain-of-replica methods involve discretising any reac-tion pathway by defining replicated conformareac-tions along
Trang 4the path between the reactants and products [32] In
order to keep the pathway along the reaction smooth
a penalty term is included into the potential function
which keeps the RMSD values equidistant between all
the neighbouring conformations [33] This modified
potential function is then used in the geometry
minimi-zation procedure to obtain the minimum energy
path-way between the reactant and product structures Since
the penalty is in the RMSD space there is no preference
in the reaction coordinate to the individual distances
among the atoms and is thus an efficient tool to
investi-gate the order of bond breaking and bond making
dur-ing the reaction process The RPATh method can be used
for both minimizations and the MD simulations In order
to investigate the basic steps of the reaction process, one
first explores the potential energy surface by minimum
energy pathway from reactants to products by a
minimi-zation procedure In the RPATh setup this means that all
the replicas are minimised along with the restraint which forces them to be equidistant in RMSD space To com-pletely understand the energetics of the reaction process one must calculate the free energy along the reaction pathways Unfortunately, it is not possible to perform such calculations with the satisfactory accuracy using the currently available computational methods, although much effort is invested to develop practically feasible QM/MM methods to calculate the free energy of a reac-tion processes [34, 35]
Study of concurrent reaction mechanisms
The following steps define the procedure for the reaction with two Mg2+ ions in the binding pocket:
1 For coordination and structural files, we read the last coordinate frame from the file produced by the 90 ns classical MD simulation as explained earlier [2]
Fig 2 Protein Tm1631, Mg2+ ions, and important amino acids in the QM region a Protein Tm1631 and b zoom in of the binding pocket used for
QM/MM calculation Protein residues that are considered as QM and 3DR7 residue of the DNA are denoted as stick models, and the two Mg 2+
ions are cyan spheres One of four link atoms is pink, others are not visible In c reactant and d product are coordinated with both Mg2+ ions (cyan
spheres), each being coordinated with 6 oxygen atoms in total The distance between O3′ and Mg22+ decreases from reactant (2.302 Å) to product (1.841 Å)
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Ogrizek et al Chemistry Central Journal (2016) 10:41
2 To perform QM/MM calculations, the QM region
and if necessary also the boundary between the
clas-sical potential and the quantum potential involving
link atoms must be defined We assigned the QM
region as follows: Lys73 side chain, two water
mol-ecules close to reaction center: water number 1863
(2.710 Å; Fig. 2 and Additional file 1: Table S1) is
involved in the reaction and nearby water 6134,
phosphate group with adjacent C5′ atom from abasic
dideoxyribose on place 7 in the DNA (residue 3DR7),
and the 5-member ring from Cyt6 residue on the
DNA We used 4 link atoms, because we divided QM
and MM region in the middle of four covalent bonds:
one in Lys73, one in 3DR7, and two in Cyt6 residues
The total number of QM atoms in the system
includ-ing two Mg2+ ions was 42 consisting of 170 electrons
described by 343 basis functions The total number of
atoms in the system was 13,932, including 2860 water
molecules
3 A reactant was constructed for which the QM/MM
minimization procedure was initiated by using RESD
[36] restraints for bonded atoms P (3DR7) and O3′
(CYT6) so that they remained separated at 1.6 Å and
for OH− (water 1863) and P (3DR7) which remained
3.5 Å apart On both bivalent metal ions, the Mg2+
harmonical restraint (the “cons harm” command in
CHARMM) was used to preserve the coordination of
ions At this stage 100 steps of ABNR minimization
were performed Then we removed CONS HARM
restraint from the minimization run of 300 steps with
only RESD restraints for bonds left Subsequently we
removed all the restraints and ran 1500 steps of
mini-mization
4 From the structure of the reactant we made a
prod-uct, breaking the bond between P (3DR7) and
O3′(CYT6) and created a bond between OH− (water
1863) and P (3DR7) 1.6 Å using restraints We also
restrained the distance between O5′ (3DR7) and
P (3DR7) at 1.6 Å After 300 steps with RESD, we
removed all the restraints and ran 1500 steps of a
geometry minimization procedure
5 We replicated the whole system 16 times and
pro-duced initial replica conformations by linear
inter-polation of the coordinates between the reactant and
the product The first and last replica represented
reactant and product conformations, respectively,
and were fixed throughout the pathway
tions With this setup a 3000 steps ABNR
minimiza-tion was performed
Procedure steps for reaction without Mg2+ ions in the
binding pocket:
1 We started with the minimized reactant from the simulation with two Mg2+ ions, then removed them from the structure and held the distance between
OH− (water 1863) and P (3DR7) at 4 Å for 300 steps Afterwards we removed all RESD restraints and ran
3000 steps of ABNR minimization
2 We took the product from the previous simulation with two Mg2+ ions and removed them; subsequently running 3000 steps of an ABNR minimization proce-dure
3 We performed an RPATh calculation, with 3000 steps
of ABNR minimization procedure using identical number of replicas as in the calculations with ions present in the system
To check the influence of the protein environment on the lowering of the energy barrier, we performed pure
QM calculations with the GAMESS program as well as the reaction path calculations with the RPATh method B3LYP/6–31G* level of theory was also used for all calcu-lations in the pure QM case
Procedure steps for reaction without Mg2+ ions and pure QM calculations:
1 First we made the reactant state from the already
Subsequently we removed the whole protein, DNA structure and water molecules that were not in the QM region Finally we ran 3000 steps of ABNR geometry minimization with restrains, holding all three protons attached to the Lys73
2 We made a product from the QM/MM calculations without Mg2+ ions in the binding pocket Then we ran
3000 steps of ABNR minimization without restrains
3 RPATh calculation was finally performed with 3000 steps of ABNR minimization, using 16 replicas
Results and discussion
The Tm1631 protein with yet unknown function from the
organism T maritima has a similar binding site to that of
DNA repair proteins, as established earlier [2] By explor-ing the possible reaction pathways usexplor-ing QM/MM meth-ods we tried to gain insight into the catalytic mechanism
of the Tm1631–DNA complex The similarity of the ener-getically most favourable pathway of the Tm1631–DNA complex with that of Mol et al [21] strongly suggests that the mechanism is the same as in other endonucleases The mechanism for Mg2+ ions catalysis that we propose
is most likely the so-called Steitz’s mechanism [23, 37]
A variety of conformations within the active site were energetically evaluated and compared and the following systems were studied:
Trang 61 QM/MM calculation for the reaction with 2 × Mg2+
ions in the binding pocket (Additional file 2: Video
S1)
2 QM/MM calculation for the reaction without Mg2+
ions in the binding pocket
3 QM calculation for the reaction without Mg2+ ions
in the binding pocket and environment with no
pro-tein or solvent molecules
Quantum mechanics/molecular mechanics and QM
calculations were performed using the B3LYP/6–31G*
DFT method As expected, obtained results suggest that
the protein has an impact on lowering the reaction
bar-rier and also establish that metal ions are required in the
binding pocket Our interest in this study is focused on
the core DNA repair function, leaving the deprotonation
of Lys73 for future investigations, however there is
evi-dence that such a state exists [8] As explained in the
pre-vious section we made sure that the ions are coordinated
as expected [38] We are aware of the possibility that one,
two or three ions may be involved in the DNA repair
reaction mechanisms However we present in this paper
the results for the two ion systems and only the core part
of the repair mechanism where the hydroxide ion attacks
the phosphate and the P-O3′ bond gets cleaved
The hydroxide ion involved in nucleophilic attack on
the phosphodiester bond P-O3′ was derived from water,
ionization of which is accomplished with the help of an
Mg2+ ion Then proton from the same water molecule
forms a bond with the nitrogen atom of the side chain
of the Lys73, which in its deprotonated state can act as
proton acceptor in the enzyme’s active site [39], and is
part of the nucleic acid repair mechanism [8] We
calcu-lated the pKa of the Lys73 to be 9.55 using the DEPTH
tool [40], which is the lowest of all lysine residues of the
Tm1631 protein We also calculated the average pKa of
the Lys73′s surrounding residues (10.5), which suggested
that Lys73 is most likely in a deprotonated state before
reaction occurs and can accept a proton from water
mol-ecule (Additional file 1: Figure S2) Literature reports that
Mg2+ plays a functional role in the catalytic mechanism
and the stability of protein-DNA complex Metal ions
also lower the local pKa, and this, considering the harsh
environment that the organism experiences, is in a good
agreement with our study [7 8 41–43]
Magnesium ions coordination is essential for most
phosphoryl transfer enzymes [44] The common catalytic
mechanism was proposed previously [23] This
mecha-nism works on the same principle as our proposed
mech-anism: Mg12+ coordinates the nucleophile and Mg22+
coordinates the leaving oxygen atom (O3′) Many similar
systems with two Mg2+ ions in the binding pocket have
been studied [12, 37] Distance between both metal ions
should be ~4 Å and in our case it is 3.85 Å (reactant) and 4.18 Å (product) Our system is also coordinated in the octahedral shape and most of the angles are 90° between O–Mg–O Also seen from other enzymatic literature enzymatic phosphate hydrolysis proceeds as SN2-like nucleophilic attack on the scissile phosphate performed
by an hydroxide ion, which is formed upon water acti-vation [24–26, 45] The important role of Mg2+ ions is lowering the pKa of its ligands and also for the presence
of second metal ion (which coordinates nucleophilic water or hydroxide in the binding pocket) This leads to a mechanism with early proton transfer [46, 47], preceding the cleavage of phosphodiester bond in case on RNase catalytic system Mg2+ ions have an essential contribu-tion for the specific catalytic reaccontribu-tions by lowering the
pKa of the leaving group and can impose specific geom-etry for the triphosphate chain—pentacovalent transition state and products [44]
We observe six critical points in the energy profile for two Mg2+ ions shown in Fig. 3 The minimum with the lowest energy is the frame 5, which is very close to
a symmetric structure, the so-called pentacovalent inter-mediate, with P atom in the center, three oxygen atoms (O1P, O2P, O5′) in a planar arrangement, and the two reacting oxygen atoms positioned almost equidistant on the opposite sides of the plane Usually such a structure would suggest a transition state in the reaction pathway but the Mg2+ ions effectively stabilize the energy of pen-tacovalent intermediate to make it a stable minimum In order to check this structure and its energy we performed
Fig 3 Energy profile (red) for each frame (1–16) of the QM/MM
calculation with two Mg 2+ ions; the values for energy are on the y1
axis Frame numbers (1–16) represent steps on the reaction path, in
which 1 is reactant and 16 is product Values for distances between atoms Cyt6 03′—P (3DR7) (green) and water 1863 OH2-P (3DR7) (blue)
are marked on the y2 axis
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Ogrizek et al Chemistry Central Journal (2016) 10:41
a separate calculation We minimized the geometry of
the single number five replica structure After 10,000
steps of ABNR minimization the geometry had no
vis-ible changes to the one in the chain of replicated
struc-tures and the energy was lowered by 2.5 kcal/mol due to
the removal of the restraint which slightly distorts the
distances and makes the energy higher The effect of the
distortion could be made smaller by increasing the
num-ber of replicas, however this would not change the
over-all properties of the reaction mechanism At the current
level of accuracy one can estimate that energy noise level
in Fig. 3 is <3 kcal/mol This makes the reaction
kineti-cally controlled exothermic and not thermodynamikineti-cally
controlled because there are no energy barriers between
the reactants and the products Our calculated energy
change (−14.0 kcal/mol) agrees with results of another
study [26], in which energy difference of
phosphodies-ter bong cleavage starting from OH− was found to be
−18.1 kcal/mol The distances to important amino acids
are reported in Additional file 1: Table S1
Next, we present the results of QM/MM
calcula-tions without Mg2+ ions (Fig. 4), which supports two
observations:
(a) There is a high 17 kcal/mol barrier in the middle of
the reaction path between structures five and nine
This structure has features analogous to those
of structure six from Fig. 3, but in this case it is a
transition state structure suggesting that the role
of the Mg2+ ions is to transform this configuration
into a stable and low energy pentacovalent
inter-mediate
(b) Many chemical reactions have multiple reaction channels which depend mostly on the positions of the species entering the reaction In the case where the two Mg2+ ions are not present the conforma-tional space of the entering species is larger than the one with the two ions present This makes the reaction energetically less favourable because the system may explore more pathways which are of higher energy than the ones with the ions present This works in addition to the fact that the environ-ment atoms in the enzyme systems usually lower the energy barriers of any transition state structure
At this point we can add that a possible solution
to choose the most favourable reaction channel would be the use of QM/MM molecular dynam-ics But there is a sampling problem to be empha-sized which, to be resolved, would require tens of nanoseconds of simulation time what translates into tens of millions steps of complete QM/MM calculations In the present studies less than 100 thousands steps of QM/MM calculations were per-formed and still it took a few months of CPU time using between 32 and 128 processors depending on the task
To show the impact of the protein, DNA, solvent and ion environment on the studied reaction, we also stud-ied the system in vacuum without Mg2+ ions The vac-uum calculation was set up keeping just the QM region
in the reaction path calculations From the Fig. 5 one can observe similar behaviour as in the QM/MM calcula-tion without ions, but the barrier is extremely high which
Fig 4 Energy profile (red) for the QM/MM calculation without ions
with the values for the energy reported on y1 axis Values for distances
between atoms Cyt6 03′—P (3DR7) (green) and water 1863 OH2–P
(3DR7) (blue) are marked on the y2 axis The distances to important
amino acids are collected in Additional file 1 : Table S2
Fig 5 Energy profile (red) for QM/MM calculation in vacuum without
Mg 2+ ions with the values for the energy denoted on the y1 axis
Values for distances between atoms Cyt6 03′—P (3DR7) (green) and
water 1863 OH2-P (3DR7) (blue) are marked on the y2 axis
Trang 8suggests that the protein environment indeed
signifi-cantly contributes to the energy stabilisation of the
pen-tacovalent transition state structure
In order to verify the stability of the initial QM/MM
setup we performed two additional classical MD 50 ns
sim-ulations starting with the reactant and product structures
that we used for the minimum energy pathway
calcula-tions The ions in these simulations kept the
hexacoordi-nated structures throughout the simulations In the case
of reactant simulations none of the waters were exchanged
and all 12 atoms around the two ions were identical This
means that the atom positions of reactant are in the stable
and favorable positions to enter the reaction
Conclusion
Protein Tm1631 from the organism T maritima was
pre-dicted to be an endonuclease-like DNA binding protein,
and consequently we investigated its function focusing
specifically on the role of Mg2+ ions in its binding pocket
We performed a QM/MM study of Tm1631 in a
com-plex with damaged DNA We found that Mg2+ ions are
required in the binding pocket in order that the reaction
occurs This allows us to conclude that Tm1631 is indeed
an endonuclease binding protein with a reaction
mecha-nism similar to that of other endonucleases Some
rec-onciliation is still needed regarding the number of metal
ions, e.g it is possible that only one ion suffices for the
reaction to take place The present paper is one of the few
theoretical insights in the available literature to study a
series of the reactions that play a role in the complex of
the endonuclease repair process Future work should be
aimed at determination of the precise number and type
of ions that are needed for the reaction to occur Another
interesting study would be to explain the formation of
the hydroxide ion in connection with the protonation of
Lys73 and the role of ions in such mechanisms It would
also be interesting to compare the present results with
the results obtained by similar studies in different
pro-teins (Additional file 3)
Authors’ contributions
The manuscript was written through contributions of all authors All authors
have given approval to the final version of the manuscript All authors read
and approved the final manuscript.
Additional files
Additional file 1. Table of distances between important atoms; RMSD
graph; pKa graph.
Additional file 2: Video S1. Movie of reaction mechanism.
Additional file 3. PDB structures for all studied mechanisms.
Author details
1 National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia
2 Laboratory for Physical Chemistry and Thermodynamics, Faculty of Chem-istry and Chemical Technology, University of Maribor, Smetanova ulica 17,
2000 Maribor, Slovenia 3 Faculty of Mathematics, Natural Sciences and Infor-mation Technologies, University of Primorska, Glagoljaška 8, 6000 Koper, Slovenia
Acknowledgements
We sincerely thank Dr Walter E Knapp for hosting Mitja Ogrizek at FU Berlin and Dr Petra Imhof for fruitful discussion.
Competing interests
The authors declare that they have no competing interests.
Funding sources
Financial support was provided through Grants P1-0002, J1-6736 and J1-6743 the Slovenian Research Agency The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Received: 6 March 2016 Accepted: 27 June 2016
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